Staphylococcal Enterotoxin B (SEB), Streptococcal Pyogenes Enterotoxin C (SPEC) and Toxic Shock Syndrome Toxin-1 (TSST-1) are among the highly incapacitating superantigen (sAg) toxins that are known to cause the incapacitating toxic shock syndrome (TSS) in human. Thus, these toxins can potentially be used in a bioterrorism attack. There is a medical need to develop broad-spectrum antibodies that can effectively be used as countermeasures. Preventing the binding of sAg toxins to the major histocompatibility complex class II (MHC-II) molecules by antagonist peptides and/or by antibodies might be an effective approach to develop such countermeasures. We have identified antagonist peptides, derived from the highly conserved domains from the sAg family of toxins. These peptides neutralized actions of SEB, SPEC and TSST-1 by preventing their binding to MHC-II molecules. Two of these peptides rendered broad-spectrum protection to mice and rabbits that were challenged with sAg toxins. More importantly, the polyclonal antibodies (pAbs) prepared against one of these peptides specifically recognized a number of sAg toxins and also neutralized their toxicities in animal models. Although the monoclonal antibodies (mAbs) against the antagonist peptide showed broad-spectrum binding to sAg toxins, but they provided only partial protection (30 to 40%) to mice challenged with SEA, raising the possibility that a single mAb might not be sufficient for the Fc-mediated rapid clearance of the toxin molecules from the body. Thus, combination of 2-3 high affinity mAbs, recognizing non-overlapping epitopes, might be necessary to provide complete and rapid protection against these toxins. Based on these studies, we have developed a novel strategy to prepare high affinity mAbs recognizing highly conserved and non-overlapping epitopes such that they can be combined to decorate toxin molecule surface at different sites for the rapid Fc-mediated clearance. Two antagonist peptides were conjugated using a meleimidepropyl ester and the conjugated peptides (c-peptides) were biotinylated. The biotinylated c-peptides were mixed with avidin at a 4:1 ratio to facilitate formation of the tetrameric c- peptides. The tetrameric antigen elicited outstanding titer of high affinity pAbs in rabbits and the generated pAbs showed broad-spectrum binding to numerous sAgs toxins on native as well as on the denaturing blots. Thus, the same tetrameric peptides antigen may also be used to immunize mice to produce mAbs. The pAbs and mAbs will be evaluated in in vitro assays and in animal models for their neutralizing efficacies. Thus, successful completion of the proposed research will lead to not only produce effective pAbs and mAbs, but also validate the utility of the c-peptides as a potential candidate for vaccination and also for intravenous immunoglobulin (IVIG) therapy. PUBLIC HEALTH RELEVANCE: Staphylococcal Enterotoxin B (SEB), Streptococcal Pyogenes Enterotoxin C (SPEC) and Toxic Shock Syndrome Toxin-1 (TSST-1) are highly incapacitating superantigen (sAg) toxins, which are known to cause toxic shock syndrome (TSS) in human. Thus, there is an unmet need to develop broad-spectrum antibodies that can be effectively used for protection against these toxins. This grant proposal is to develop high affinity polyclonal and monoclonal antibodies that can be used as broad-spectrum antidotes against sAg toxins. The proposed research will also validate a novel approach to use the avidin/biotin- tetrameric peptides formulation as an antigen to produce antibodies against small peptides, and also validate its utility as a potential candidate for vaccination and/or for intravenous immunoglobulin (IVIG) therapy.